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Anti-sperm antibodies
Sperm antigenicity within the animal kingdom was first described in 1899. In 1932, Baskin observed circulating antibodies against sperm and finally in 1954, Rümke observed and described the first type of anti-sperm antibodies. ASA have cytotoxic, immobilizing and agglutinating (Fig. 10) functions. The sperm-agglutinating type is most abundant among the European population and causes the so-called shaking phenomenon, while the sperm-immobilizing type is more prevalent in the Asian population. ASA are detectable on the systematic (blood and lymph) as well as the local level (SF, cervical-vaginal mucus). In general, the IgG isotype of ASA is mainly related to blood circulation and the IgA isotype to mucosal immunity in women. In men, IgG and IgA fractions are most prevalent in SF, while IgG and IgM isotypes prevail in serum (Ulcova-Gallova, 2006).
Sperm carries the paternal genome further displayed on an oocyte and has a very heterogeneous antigenic content. Since sperm has auto-antigenic (auto-immunization) as well as iso-antigenic (iso-immunization) potential, it is capable of inducing the production of sperm-reactive T-cells in men as well as in women. It is not a single ASA that influences fertility, but more likely an ensemble of ASA that causes infertility. It has been postulated that a single sperm antigen is not able to cause infertility. Further, it has been reported that not all ASA, regardless of being produced in women or men, influence the fertility potential since the cognate antigen is not necessarily involved in the fertilization process (Wang et al., 2009; Haidl, 2010; Sedlackova et al., 2010; Bronson, 2011). A highly heterogeneous sperm antigenic content could even be modified during maturation and ejaculation based on antigen sequestration. Newly expressed antigens could be then in contact with any immunocompetent cells. For example, a sperm membrane-incorporated fibronectin exhibits changes in regional antigenic expression during sperm maturation, whereas secreted fibronectin is a product of male accessory sex glands and can be attached to sperm tails during ejaculation (Kosanovic and Jankovic, 2010). Considering gastrointestinal exposure, ASA formation may be operative (Bohring and Krause, 2003).
In men, sperm germ cells are protected in the testis from an auto-immune attack by the blood-testis barrier. When the barrier is disrupted, ASA are detectable in blood serum, seminal plasma or directly attached to sperm surface membrane. An increased risk of ASA formation may follow the congenital absence of reproductive tract components. ASA are mostly associated with genital inflammation or infection (e.g. orchitis), epididymis trauma, genital surgery, cryptorchidism and varicocele (Kosanovic and Jankovic, 2010). The theory of auto-immune disease was supported by Omu et al. (1999) by proving that ASA formation is related to certain human leukocyte antigen (HLA) classes.
Association of seminal components with female sensitization
It has been shown that SF, a nutritious transport buffering sperm medium, has a built-in mechanism for preventing an immunological sensitization of the female. This protective system exists due to the presence of immune inhibitors originating in the male sex accessory glands (Prakash, 1981). Some seminal constituents, such as cathepsin D, are able to degrade vaginally exposed proteins that may be involved in antibody formation related to immune infertility. Furthermore, the low molecular weight proteins are of need in oocyte and sperm fusion as degradation products (Pardesi et al., 2004). Seminal ZAG, a 40 kDa multidisciplinary protein, has been reported as a novel adipokine playing a significant role in fertilization, lipid mobilization, and peptide/antigen/ligands binding. ZAG may participate in the expression of female immune response since the fold is similar to major histocompatibility complex (MHC) I antigen-presenting cells. ZAG has proved to be an IgG-binding protein related to a pathophysiological iso-immunization. It may also have a protective character, as it belongs to the immunoglobulin gene superfamily and thus blocks the newly created female anti-semen antibodies (Hassan et al., 2008; Brazdova et al., 2013b). An antibody fraction interacting with a seminal antigen targets most of seminal proteins adsorbed on sperm. However, SF induces the recruitment of macrophages and dendritic cells into cervical and endometrial tissues. SF has been heavily involved in the rare IgE-mediated reaction to semen (Weidinger et al., 2006; Brazdova et al., 2012a).
Auto-immune aspects in infertility
Auto-immune phenomena have been previously associated with an increased prevalence of female immune infertility. This finding names anti-phospolipid, anti-nuclear, anti-thyroid, anti-annexin V, anti-protombin, anti-laminin, anti-zona pellucida antibody formation, as well as a high level of NK cells as the risk factors but not as those pathognomonic (Gleicher and Barad, 2006).
ZP, as the protective layer, is composed of glycoproteins. It represents a broad antigenic content. Antibodies against ZP prevent sperm from penetrating it. Anti-ZP autoantibody concentration can be elevated if the shape of ZP is abnormal (deformed, thickened, or thinned). These antibodies interfere with the implantation process since ZP protects the fertilized oocyte up to the 7th day after fertilization and until embryo hatching. ZP is, during this time, thickened. ZP-specific antibodies are detectable in follicular and peritoneal fluid and cervical mucus in IgG, IgA and IgM isotypes (Ulcova-Gallova, 2006).
Anti-phospholipid antibodies (APA) were first found by Wasserman in 1906 and since that time have been associated with miscarriage, intrauterine fetal death and placental thrombosis. These components of the female immune system are autoantibodies directed against the negatively charged parts of phospholipids, and in particular phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, annexin V, cardiolipin, and 2-glycoprotein. APA are mostly produced in the IgG fraction accompanied by IgA and IgM. Phosphatidylserin-specific APA cause fetus hypotrophy as a consequence of placental vascular damage. However, mother protection results in the higher production of anti-coagulating factors. Furthermore, the risk of spontaneous abortion is higher in the presence of anti-coagulating antibodies. Antibodies specific to annexin V and placental anti-coagulating protein are risk factors in reproductive failure. These IgG antibodies are detectable in 5-6% of women diagnosed with pregnancy loss, in 8-10% of women after unsuccessful in vitro fertilization (IVF), in 1% of not pregnant and healthy women and in 0% of pregnant women without pathophysiologic aspect. Complex complication is called anti-phospholipid syndrome (APS), also known as Hughes syndrome. It may cause hyper-coagulation leading to rapid organ failure. Since APA have high affinity to phosphatidylserin present on trophoblasts, APS seems to be involved in the mental retardation of newborn children (Ulcova-Gallova, 2006; Ruiz-Irastorza et al., 2010). The trophoblast expresses the major histocompatibility antigens on its surface. They are of maternal and paternal origin. Paternal patterns induce allo-immunity. Formed anti-paternal cytotoxic antibodies usually implicate spontaneous abortion. Other factors involved in this complicated immune reaction are NK cells, alteration in Th1 and Th2 ratios and abnormal HLA-G expression (Shetty and Ghosh, 2009).
Endometrium-specific antibodies are inter alia associated with the polycystic ovary syndrome (PCOS) that is mainly classified as an endocrine genetic disorder. Women suffering from PCOS have usually problems getting pregnant. PCOS is known as Stein-Leventhal syndrome described in 1935. It is characterized by enlarged ovaries caused by cysts, irregular ovulation, irregular or no menstrual periods and increased androgen level. With regards to androgen level, PCOS is associated with hirsutism. On the other hand, it is associated with obesity, type 2 diabetes and high cholesterol level (Ulcova-Gallova, 2006; MFMER, 2013).
Pregnancy is complicated by a serious gynecological complication called endometriosis. It affects up to 10% of women in reproductive age and 25% of women diagnosed with endometriosis are infertile. Its etiology is unknown. This disorder occurs when the lining of the uterus grows in other areas outside of uterus. Ectopic bearings react on the hormone level, thus are subject to the menstrual cycle. Created cysts occur on ovaries, fallopian tubes, peritoneum, cervix and in vagina. Peritoneal endometriosis is characterized by retrograde menstruation causing secondary inflammation. Factors typical for such a condition are the high level of autoantibodies, presence of T-lymphocytes in peritoneal fluid and elevated level of NK cells (Nisolle et al., 1990; Bulletti et al., 2010).
Mucosal immunity of the female genital tract
The mucosal immune system operates on a local level and is represented by lymphoid tissues in mucosae and external secretory glands. It limits the access of environmental antigens, by which the fertility potential is significantly regulated as well. Any inert antigen is attacked by these secreted agents of protection. It restricts and/or avoids penetration in the systematic compartment. The female genital tissues and secretion (vaginal washes and cervical mucus) provide protection that differs from systematic reactions by the cell types involved and by their products, antibodies. However, it is the initial antigen exposure to mucosae that leads to the systematic T-cell hypo-responsiveness (Woof and Mestecky, 2005; Mestecky et al., 2007).
Mucosal immunity in the female genital tract is influenced by the level of antibodies, cytokines and hormones. Humoral immune defense displayed in musocal tissue surfaces is comprised of the IgG, IgA and IgM antibody isotypes. IgG, IgA and IgM levels are dependent on the menstrual cycle, and so are influenced by hormones. IgA and IgG reach maximum concentration before ovulation, which is linked to an increased level of interleukin 1 component (IL-1 ). Particularly, estrogen causes a higher expression of secretory IgA (S-IgA), thereby increasing the rate of selective transport. This method of regulation is responsible for antibody-isotype distribution and the characteristics thereof, including their individual properties and the transport of immunoglobulin-containing cells, antigen-presenting cells (APC), CD4+ and CD8+ cells in the vagina, uterus and fallopian tubes (Kutteh et al., 1996). In addition, it has been shown (Franklin and Kutteh, 1999) that oral contraception influences IgA as well as IgG populations in cervical mucus. It is around one third larger than in the cervical mucus of naturally cycling women. Further, the vaginal washes of women on oral contraception present an elevated level of IgG in comparison to IgA. Several observations showed (Kutteh et al., 1996; Mestecky and Fultz, 1999) that the concentration of these antibodies decreases as IgG>IgA>IgM. This relationship is linked to the presence of IgG/A/M-producing cells. The uterine endocervix contains the highest amount of IgG- and IgA-secreting cells. Cervical mucus contains higher levels of IgG than IgA, both of which are locally produced. By contrast, women on oral contraception have IgA as the predominant antibody present in cervical mucus. Among the mentioned three isotypes, IgM is the least efficiently transported antibody. The mucosal IgA antibodies are selectively transported for external secretion based on a receptor-associated mechanism. The distribution of IgG subclasses in mucosal secretion displayed concentrations proportional to that of plasma. IgD occurs rarely or in very low concentration in external mucosae. The level of IgE depends on the allergenic nature of presented antigen (Woof and Mestecky, 2005).
Despite the low IgA affinity, the avidity is high regarding multi-binding sites. Proteolytic enzymes usually degrade environmental antigens leading to a relatively low mucosal absorption rate. IgA itself is resistant to enzymes of proteolytic character. It has been suggested that certain amounts of non-eliminated antigens circulate in the complex with IgA, which further activates the systematic immune response. It is less probable that an antigen only just entering the mucosal tissue could circulate on its own (Mestecky and Fultz, 1999; Mestecky et al., 2007; Corthesy, 2007).
IgA is a multivalent antibody existing in two subclasses, IgA1 and IgA2. While IgA1 responds to viral (protein) antigens, IgA2 binds to lipopolysaccharide or polysaccharide structures. IgA has an anti-inflammatory activity evidenced by the inhibition of complement activation and by a diminishing effect on NK cells. These properties might inhibit an early and precise diagnosis as no inflammatory marker may be detected. In cervical mucus as well as vaginal washes, IgA1 concentration is equal to that of IgA2. S-IgA is locally produced by sub-epithelial plasma cells. Most of the time, it is a polymeric molecule that corresponds with IgA2 since IgA1 is rather monomeric. It has been suggested that cervical mucus contains about 80% of the polymeric form and vaginal wash about 50% (Kutteh and Mestecky, 1994; Kutteh et al., 1996; Woof and Mestecky, 2005). The IgA antibody can degranulate eosinophils that cover mucosal surfaces. This pathology is observed when natural immune tolerance is disrupted. (Further reaction may evoke allergic reaction to the presented antigen such as seminal and/or sperm structure. Semen rejection at the level of mucosal immunity does not have to be displayed on the systematic level (Woof and Mestecky, 2005).
Woof and Mestecky (2005) discussed the protective character of IgA. Its protective role is observed in an antibody-dependent, cell-mediated cytotoxicity, opsonization, the activation of innate humoral factors, and the removing and further elimination of already formed immune complexes within epithelial cells and lamina propria. It is known that IgA is able to diminish the partial and total absorption of the antigen on mucosal tissues. In comparison with IgG, which after antigen-recognition activates complement resulting in inflammation, IgA acts as an inhibitor to directly avoid the adherence of antigen (Russell et al., 1997). S-IgA in a complex with antigen is not able to efficiently activate the complement pathway. Exclusivity of IgA is related to its natural occurrence and polyreactivity. Another characteristic of its protective capacity is related to the cell line that produces the IgA subclasses. IgA1 originating in B1 cells are of low affinity but wide specificity. In contrast, S-IgA derived independently from B1 and B2 cells do not differ in antigen specificity (Quan et al., 1997).
Cervical Mucus
The uterine cervix participates in the local immune reaction by the application of immunoglobulin-producing cells in a complex mixture known as cervical mucus/fluid/plasma, which is located in and around the cervix. Cervical mucus is composed of up to 90% water, depending on the menstrual cycle, which determines the quantity as well as the quality of cervical mucus. Its composition is based on a glycoprotein web filled with mucus rich in immune-competent proteins, electrolytes (calcium, sodium and potassium), simple sugars such as fructose and glucose, amino acids, C3 and C4 complement components, Th1 and Th2 cytokines, the prostaglandins of E series, and trace elements (zinc, copper, iron, mangan, selenium). An imbalance is frequently associated with immune infertility and spontaneous abortion (Moghissi, 1972; Schumacher, 1988; Cibulka et al., 2005; Ulcova-Gallova, 2010).
Optimal pH ranges from 7.4 to 9.6. The pH of cervical mucus is alkaline during ovulation to allow sperm survival due to elevated levels of water and electrolytes. After menstruation, cervical mucus becomes rather acidic. Acidic pH is characteristic for vaginal mucus as well, ranging from 3.7 to 4.5 (Gruberova et al., 2006).
The basic role consists of a barrier that prohibits anything from entering the uterus. It is associated with “stick and thick” properties. Cervical mucus acts as a natural lubricant due to its glycerol content. The apparent amount of cervical mucus is not hormone-dependent. The mucus also functions as a transport and nourishing medium for sperm by being less concentrated, transparent with a lower amount of immune-competent agents and a high fructose level, which is essential for an efficient sperm metabolism. The sugar level is progesterone dependent. On the other hand, the cervix always acts as a reservoir for sperm after sexual intercourse. Regarding iso-immunization during the entire menstrual cycle, cervical mucus contains the antibodies directed toward sperm. Their amount is then crucial for sperm-cervical mucus penetration and following fertilization. ASA-positive female patients have been commonly diagnosed with immune infertility. Approximately 5-10% of infertile female patients are positive in cervical ASA. An iso-immunization rate is observed by probing the local ASA level, which will determine an appropriate treatment (Moghissi, 1979; Schumacher, 1988; Cibulka et al., 2005). It has been shown (Ulcova-Gallova, 2010) that ASA present in cervical mucus are of agglutinating character. These locally produced ASA do not differ from those systematically produced thus they affect sperm capacitation, acrosome reaction, and may interfere with zona pellucida penetration as well as embryo implantation (Livi et al., 1991). The peak of ASA in IgA and IgG fraction is reached at the luteal and follicular phases of the menstrual cycle. In contrast, their levels are lowest during ovulation. This peak in ASA concentration is related to the highest level of estradiol, usually observed one day before ovulation (Franklin and Kutteh, 1999). However, Schumacher (1973) demonstrated that local immunoglobulin production could be diminished in women on oral contraception. The study demonstrated the impact of oral contraception with respect to estrogen-dependent, decreasing antibody level in cervical mucus and then with regards to progestin-dependent (a synthetic progestogen of progestational effects similar to progesterone), increasing antibody level (Schumacher, 1973). This finding supports the fact that cervical immunity before ovulation is influenced by estrogen and after by progesterone.
IVIg and infertility related disorders
IVIg may represent a potential therapeutic strategy in fertility disorders related to an immune system with an auto-immune background. SLE is considered as the most serious disorder related to infertility. It is a multifactorial auto-immune disease characterized by the presence of autoantibodies linked to nuclear antigens. It is characterized by system failure that can affect any part of the body. Created immune complexes precipitate. This action further activates an immune response. Since SLE is associated with fetal death or miscarriage, treatment by IVIg might be of potential interest. Unfortunately, sufficient randomized studies to prove the positive effect of IVIg, as has been done for previously mentioned diseases, are few in number (Ulcova-Gallova 2006; Smyth et al., 2010; Bayry et al., 2011). Pregnancy complication and pre-eclampsia may be related to anti-phospholipid syndrome. Anti-phospholipid and antiapolipoprotein antibodies provoke thrombosis. Spontaneous abortion can be prevented by IVIg dose of 1g/kg per day for 2 consecutive days every month until birth. As the study of Coulam and Acacio (2012) showed, IVIg treatment did not bring any benefit in comparison with combined therapy involving heparin and aspirin. It has been speculated whether or not the advantage of IVIg administration profits from a blockade of the neonatal IgG-Fc receptor. It may lead to the catabolism of pathologic antibodies. Anti-cardiolopin antibodies could be then neutralized by anti-idiotypic antibodies (Bayry et al., 2011; Coulam and Acacio, 2012).
Disintegration of human sperm and characterization of its antigen
The active and sensitized immune system may affect fertility. Immunological background has been studied in association with sperm antigens. A change in cellular immunity against sperm may cause the antibody-mediated rejection of semen. Our paper represents the first part of research that deals with the immunological properties of sperm concerning female immune infertility. Despite the progress in molecular genetics and proteomics, we consider sperm disintegration to be a key step in sperm antigen characterization. Regarding post-translational modifications that seem to be essential for efficient spermatozoa, it is necessary to prepare the protein extract in order to keep as many conformational and linear epitopes on the sperm antigens as possible. On the other hand, not all processes of sperm disintegration meet these conditions. Then, several epitopes or entire patterns might be lost or damaged.
In this study, we focused on the mechanical and chemical disintegration in order to compare the detected particular sperm antigens and antigenic patterns recognized by immunoblotting. The sperm samples were processed by sonication, hypo-osmotic shock and utilization of detergents, urea and benzalkonium chloride, to process the sperm disintegration concerning the above mentioned conditions. The source of antibodies was the sera of female patients diagnosed with fertility failure. Sera were collected according to the level of sperm-agglutinating antibodies using the Fridberg test and antibody class using indirect MAR test. A negative control was required as well. A serum of 10-year-old girl was apparently appropriate since it had been presumed that ASA might not have been present. The complex repertoire of sperm antigens was detected in the sperm protein extract that was prepared using Triton X-100. The most frequent sperm antigens had 68 and 123 kDa.
Table of contents :
1 INTRODUCTION
2 MALE FACTOR IN REPRODUCTION
2.1 Seminal fluid
2.2 Spermatozoa
2.2.1 Spermatogenesis
2.2.2 Capacitation
2.2.3 Acrosome reaction
2.2.4 Fertilization
3 FEMALE FACTOR IN REPRODUCTION
3.1 Oocyte
3.1.1 Oogenesis
3.2 Embryo development
4 INFERTILITY
4.1 Unexplained infertility
4.2 Immune infertility
4.2.1 Anti-sperm antibodies
4.2.2 Association of seminal components with female sensitization
4.2.3 Auto-immune aspects in infertility
4.3 Mucosal immunity of the female genital tract
4.3.1 Cervical Mucus
4.4 Treatment of female infertility
4.4.1 Fertility drugs
4.4.2 Reproductive assistance
4.4.3 Intravenous immunoglobulins
4.4.3.1 IVIg and infertility related disorders
5 RESULTS
5.1 Publications
5.1.1 Anti-sperm antibodies
5.1.2 Disintegration of human sperm and characterization of its antigen
5.1.3 IgG, IgA and IgE reactivities to sperm antigens in infertile women
5.1.4 Female serum immunoglobulins G, A, E and their immunological reactions to seminal fluid antigens
5.1.5 Immunodominant semen proteins I: New patterns of sperm proteins related to female immune infertility
5.1.6 Immunodominant semen proteins II: Contribution of seminal proteins to female immune infertility
5.1.7 Immunodominant semen proteins III: IgG1 and IgG4 linkage in female immune infertility
5.1.8 Pre-eclampsia: a life-threatening pregnancy syndrome
6 FUTURE ASPECTS
6.1 Design of a miniaturized diagnostic tool
6.2 Immuno-Intervention in female immune infertility
7 DISCUSSION
7.1 Antibody recognition
7.2 Protein markers
7.3 Immunoassay to screen female semen sensitivity
7.4 Immuno-Intervention strategies
8 CONCLUSION
9 PERSPECTIVES
10 REFERENCES
11 LIST OF FIGURES
12 LIST OF TABLES
13 LIST OF ABBREVIATIONS
14 ANNEXES
14.1 Publications not related to the topic of female immune infertility
14.1.1 Tumor markers and their use in clinical practice
14.1.2 Indoor long-term persistence of cypress pollen allergenic potency: a 10month study
14.1.3 Complementarity between microarray and immunoblot for the comparative evaluation of IgE repertoire of French and Italian cypress pollen allergic patients
